The development of the first anti-infective or anti-bacterial agents in the early 1900's and the success seen with their use led to the development of hundreds of new compounds to fight the wide variety of bacterial organisms. Most people have had exposure to these drugs at some point in their life, with the majority of situations resulting in a rapid cure of the bacterial infection with relatively few side effects. However, human morbidity and mortality due to bacterial infections has become a major concern today due to the dramatic increase in the frequency of infections caused by bacteria that are resistant to most, if not all, of the available anti-bacterial agents. This increase in drug-resistant pathogens has led to an ever-increasing need for new drugs with new mechanisms of action.
Anti-bacterial agents are developed by identifying unique targets not present in mammalian cells and then designing a drug to exploit that difference such that the bacterial cells are killed or neutralized while mammalian cells are left intact and unaffected. The goal of successful anti-bacterial drug therapy is to limit toxicity in the patient while maximizing the ability of the drug to invade the bacterial cells and neutralize those cells as selectively as possible. The major classes of anti-bacterial drugs available today target a variety of different cellular components and functions of bacteria such as the cell wall, protein synthesis, cell metabolism, DNA synthesis, and the bacterial cell membrane. Each of these target cellular components or functions is related in some way to the disease process of bacterial infections that involves first colonization of the bacteria, invasion of host cells, production of cellular toxins or inflammatory agents, and a host response to those toxins or agents.
A fundamental process of all living cells, including bacteria, is the secretion of proteins across membranes. The majority of proteins that are secreted are synthesized as a precursor with an N-terminal signal sequence (or leader peptide) of about 16-24 amino acids in length. This leader sequence serves to promote recognition of the protein by the secretory apparatus of the cell and facilitates movement across the membrane. The leader sequence is subsequently processed by a leader peptidase to remove the sequence and allow release of the mature or active protein. Recent research has indicated that in the case of bacteria, there are several systems for secreting proteins and some of these systems have unique leader peptidases associated with their cognate secreted proteins. One of these systems is known as the type 2 secretion system which promotes extracellular secretion of bacterial factors such as toxins and colonization pili that are the hallmarks of the mechanisms that promote virulence of pathogenic bacteria. Pili mediate the binding of bacteria to host tissues and most pili are composed of a major protein subunit that polymerizes to form a pilus.
The type 2 secretion systems of most bacteria involve a type 4 pilin for colonization pilus formation and type 4 pilin-like proteins for secretion of toxins and other factors associated with bacterial virulence and destruction of host tissue and enhancement of bacterial growth in the host. Highly related type 4 pili serve as the major colonization factors for up to 50 different gram-negative bacterial species and type 4 pilin-like proteins have been found for a growing number of gram-positive bacteria as well. Type 4 pili are composed of a polymerized structure of type 4 pilin. The pilin is synthesized as a prepilin with a leader peptide that is very different from those of typical secreted proteins. A type 4 specific leader peptidase is required to process a type 4 prepilin leader sequence to allow secretion of the mature protein. Importantly, this secretion system including the type 4 leader peptidase itself is only found in bacteria and is not present in humans or other potential hosts of infection. Furthermore, mutating the type 4 prepilin peptidase (TFPP) renders the bacterium avirulent (March and Taylor 1998. Mol. Microbiol. 29:1481-1492).
The type 4 signal peptide is highly conserved across all type 4 prepilin or prepilin-like proteins and is composed of 6 to 25 highly charged amino acids at the N-terminus followed by approximately 20 predominately hydrophobic amino acids. Cleavage occurs between the two domains immediately C-terminal of an invariant glycine and before the new N-terminal amino acid that is usually a methionine or a phenylalanine. Unlike cleavage of standard signal peptide by signal peptidase I, wherein the cleavage occurs on the periplasmic side of the inner membrane, processing by a type 4 peptidase occurs on the cytoplasmic side of the inner membrane (Strom and Lory, 1993. Ann. Rev. Microbiol. 47:565-596). Previous mutational analysis and protease inhibitor evidence from studies of pilD of Pseudomonas aeruginosa and protein alignment analysis of the type 4 peptidase family suggested two pairs of cysteines in cytoplasmic domain 1, the largest cytoplasmic domain, to be involved in the protease active site of the enzyme (Strom et al. 1993. Proc. Natl. Acad. Sci. USA 90:2404-2408). These data resulted in the categorization of type 4 prepilin peptidase (TFPP) family as a type of cysteine protease (Strom et al. 1994. Meth. Enzymol. 235:527-540).
However, subsequent studies have shown the two cysteine pairs to be lacking in XpsO, a type 4 prepilin peptidase of X. campestris. In fact, several type 4 prepilin peptidases, among the approximately 27 that have been identified and cloned, do not have the conserved cysteines in their protein sequence but still function to cleave the type 4 signal peptide.
In the present invention a specific domain of type 4 prepilin peptidase that is essential for cleavage activity has now been identified to the resolution of two specific amino acids. Identification of this domain has facilitated the identification of specific inhibitors of the protease activity of type 4 prepilin peptidase as well as other TFPP-like aspartyl proteases which utilize this same cleavage mechanism.